6518811Broad Institute2138-03_Inhibitor_Dose_CherryPick_Activity2012128Inhibition of T. cruzi Tulahuen strain amastigotes in L-6 muscle cells Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2138-03_Inhibitor_Dose_CherryPick_ActivityKeywords: T. cruzi, Tulahuen, amastigotes, Trypanosoma cruziAssay Overview: Rat skeletal myoblasts (L-6 cells) were infected with T. cruzi Tulahuen strain C2C4 containing the a-galactosidase (Lac Z) gene (Buckner et al. 1996). After 48 hr, extracellular T. cruzi were removed and cells were treated with or without compounds. After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. a-galactosidase, and thus T. cruzi proliferation, was measured by additon of TCPRG/Nonidet. Wells were read photometrically at 540 nm within 2-6 hrs of read reagent additon. Expected Outcome: Compounds significantly suppressing CPRG colorometric assays, and therefore a-galactosidase expression will be identified as hits in the screen. Compounds that inhibit color development of CPRG or are toxic to the host cell will also be identified as actives. Protocol:Assay Description: Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtitre plates at 2000 cells/well in 100 iL RPMI 1640 medium with 10% FBS and 2 mM l-glutamine. After 24 h the medium was removed and replaced by 100 il per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the a-galactosidase (Lac Z) gene (Buckner et al. 1996). After 48 h the medium was removed from the wells and replaced by 100 il fresh medium with or without a serial drug dilution of eleven 3-fold dilution steps covering a range from 100 to 0.002 ig/ml. After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then the substrate CPRG/Nonidet (50 il) was added to all wells. A color reaction developed within 2-6 h and could be read photometrically at 540 nm. Data were analyzed with the graphic programme Softmax Pro (Molecular Devices), which calculated IC50 values by linear regression (Huber 1993) from the sigmoidal dose inhibition curves. Benznidazole is used as control.Standard assay parasite strains: T. cruzi Tulahuen C2C4, containing the Lac Z gene.Plasmid construct by Dr. S. ReedReceived from Dr. Buckner, UW, Seattle as epimastigotes in LIT, transformed by E.G.Standard Cell line:L-6 cells (mouse muscle fibroblasts)Standard Drug:Benznidazole (RadanilTM, Hoffman La Roche): start conc. 30microg/ml (IC50 = 0.35 microg/ml)Standard Conditions:Medium: RPMI 1640 + 10% FCS + 1.7microM L-Glutamine (850microl 200mM for 100ml)Plates: CostarTM 96-well microtiter platesIncubation: 370C, 5% CO2Substrate: 2.5X CPRG/Nonidet Solution: 5X stock = 500 ml Nonidet P40+ 30.38mg CPRG in 100 ml 1X PBS Dilute the 5X stock 1:1 with 1X PBS. Light Sensitive!Definition of test score: inactive (no repeat): IC50 > 30mg/mLmoderate activity (repeat): 2mg/mL < IC50 < 30mg/mLhigh activity (repeat): IC50 < 2mg/mL (active series < 1mg/mL)Drug preparation: Compounds are dissolved in DMSO at 10mg/ml (SOP Nr. D1). If insoluble other solvents are used according to the recommendations of the supplier. The DMSO stocks are kept at -20 degrees C. For the assays fresh dilutions in medium are prepared each time. (Since DMSO is toxic, care has to be taken not to exceed a final concentration of 1% DMSO in the assay).Procedure:Day 1:Seed all 96 wells with 100 microl medium containing 2x103 L6 cells per well, using a multi-well repeater pipette. 9.6 ml per plate 2x104 L-6 cells/ml....100 microl per wellDay 2: Add 5x103 tryps into all columns 1 and 2, 4 and 5, 7 and 8, and 10 and 11 using the multi-well repeater pipette. In columns 3, 6, 9 and 12, add 50 microl of medium. 3.2 ml of trypanosome suspension per plate 1x105 tryps/ml.......50 microl per wellDay 4:Remove medium from wells in row A to G with the aspirator and replace with 100microl medium using the multi-well repeater pipette. (Take care to not cross infect columns 3, 6, 9 and 12-remove medium only before medium + tryps!)Remove medium from row H (do not cross-infect!) and replace with 150 microl of the highest drug concentration. Per plate 4 drugs can be tested (drug 1 columns 1-3, drug 2 columns 4-6, etc.). Note: Do the first half of the plates (remove medium and add drug) and then the second half, so the cells/tryps don't dry out.Serial drug dilutions are prepared by using a 12-channel multi-pipette. First, remove 50 microl from wells of row H and put into row G and mix well. Next, 50 microl are taken out of row G and put into row F and so on until row B. The last 50 microl of row B are discarded. A serial dilution factor of 1:3 is thus obtained. Wells in row A serve as control wells without drugs.Day 8: Evaluate the plates visually to determine the MIC (Minimal Inhibitory Concentration): lowest drug concentration at which no trypanosomes with a normal morphology and motility as compared to the control wells can be seen. Additional information may be recorded, such as drug insolubility or contamination, etc. Also, preliminary cytotoxicity (MIC) is to be noted in columns 3, 6, 9 and 12.50 microl of 2.5X CPRG/ Nonidet are added to all wells. A color reaction will become visible in 2-6 hours and can be read in an Absorbance Reader at 540nm.Data are transferred into a graphic program (e.g. Excel) and IC50 values are calculated.EXPECTED OUTCOME: Compounds with IC50 less than 25 fold the control compound, Chloroquine, were labeled active.624280Summary assay19238193Identification of Three Classes of Heteroaromatic Compounds with Activity against Intracellular Trypanosoma cruzi by Chemical Library Screening8913471Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase85057Beta-galatsidase expressing (Tulahuen) strain created and described in article: PMID8913471http://www.broadinstitute.orgBroad Research Institute1IC50_uM15221 R03 MH085673-01285792654Broad InstituteBRD-K38553273-001-01-7112912.95201212685793296Broad InstituteBRD-K50941930-001-01-2111018.41201212685793830Broad InstituteBRD-K02108475-001-01-8111415.91201212685795059Broad InstituteBRD-K42349777-001-01-5112213.79201212685795088Broad InstituteBRD-K52072667-001-01-3111217.39201212685797392Broad InstituteBRD-K81391936-001-01-7111515.59201212685799144Broad InstituteBRD-K64366758-001-01-5127411.15201212685800201Broad InstituteBRD-K82092559-001-01-6128311.02201212685800744Broad InstituteBRD-K09344309-001-01-4111515.79201212685801692Broad InstituteBRD-K28800414-001-01-4113112.78201212685801999Broad InstituteBRD-K32625677-001-01-1127111.2201212685802019Broad InstituteBRD-K74155249-001-01-7111515.64201212685802226Broad InstituteBRD-K24861700-001-01-3112613.22201212685802685Broad InstituteBRD-K67798612-001-01-2124711.82201212685803077Broad InstituteBRD-K14340219-001-01-4111814.77201212685803477Broad InstituteBRD-K43806450-001-01-7111814.83201212685803906Broad InstituteBRD-K60359902-001-01-9127511.14201212685805371Broad InstituteBRD-K99381377-001-01-4114119.2201212685806902Broad InstituteBRD-K21786893-001-01-2114120.6201212685807205Broad InstituteBRD-K62499920-001-01-9112613.212012126